The present invention relates to real-time or near real-time detection in gases or fluids of nerve agents of the sort commonly encountered in chemical warfare agents.
Often referred to as the xe2x80x9cpoor man""s nuclear weaponxe2x80x9d, chemical and biological weapons of war are so named because they cost much less than real nuclear weapons to develop, do not require a high level of technology to produce, and can potentially kill enormous numbers of people. Indeed, unlike nuclear weapons, which require a large, specialized, and costly scientific-industrial base, chemical and biological agents can be made with commercial equipment generally available to any country. Weapons of this sort are especially attractive for use by developing countries against super powers, as they tend to level the playing field in struggles against these better armed and trained opponents. The use of biological and chemical weapons of mass destruction is banned by international treaty, but reports of suspected and confirmed use continue.
Biological weapons can be produced from widely available pathogens which may be procured for legitimate biomedical research or obtained from soil or infected animals and humans. Moreover, many of the infectious diseases associated with biological warfare are endemic to most of the states suspected of developing a biological weapon capability. Biological agents are thus both cheap and easy to obtain: in effect, any nation with a basic pharmaceutical industryxe2x80x94or even a facility such as a breweryxe2x80x94has the capability of producing biological weapons.
Biological agents contain either living organisms or their derivatives, such as toxins, which cause disease or death in humans, animals, or food crops. Living organisms multiply within the living targets to produce their effects, whereas toxins cannot reproduce themselves. Toxins are generally more lethal, and act relatively quickly causing incapacitation or death within minutes or hours. Living organisms (microbial pathogens), require incubation periods of from 24 hours to 6 weeks between infection and appearance of symptoms. This incubation period places limits on their battlefield utility, but it also means that biological weapons can continue to have a significant impact many weeks after the initial attack (e.g., by causing a long-term pandemic). Likewise, this delayed incubation period may mean that a biological attack can be completed before those on the ground have realized that it has occurred, or even take place entirely covertly, the effects being confused with a natural outbreak of disease.
Biological agents are odorless, tasteless, and when dispersed in an aerosol cloud, are invisible to the human eye because the particle size of the aerosol is extremely smallxe2x80x94as small as 1 to 5 micrometers or microns. Weight-for-weight, biological weapons are hundreds to thousands of times more potent than the most lethal chemical weapon, meaning that even small amounts (e.g., a few kilograms) could be used with devastating effect, whereas hundreds or thousands of tons of chemical agents could be required for militarily significant operations.
Among lethal chemical warfare agents, nerve agents have played a dominant role since the Second World War. Nerve agents are so-called because they affect the transmission of nerve impulses within the nervous system. Nerve agents belong chemically to the group of organo-phosphorus (xe2x80x9cOPxe2x80x9d, hereinafter) compounds. OP compounds are stable, easily dispersed, highly toxic, and take effect rapidly both when absorbed through the skin and via respiration. They can be manufactured by means of fairly simple chemical techniques and the raw materials to manufacture them are inexpensive and generally readily available. Sarin, one of the more familiar nerve agents, dates from the Second World War and is considered a xe2x80x9cclassicxe2x80x9d substance. In the mid-1950""s, however, a group of more stable nerve agents known was the V-agents were developed, with VX being one of the more successful variants. These later-day chemical weapons are approximately ten-fold more poisonous than sarin and are thus among the most toxic substances ever synthesized.
Nerve agents in pure state are colorless liquids with volatiles that vary depending on the particular compound. The consistency of VX may be likened to a non-volatile oil and is therefore classified as belonging to the group of persistent chemical warfare agents. It enters the body mainly through direct contact with the skin. Sarin is at the opposite extreme, being a relatively volatile liquid (comparable with, e.g., water), and is mainly taken up through the respiratory organs.
The nerve agent, either as a gas, aerosol or liquid, enters the body through inhalation or through the skin. Poisoning may also occur through consumption of liquids or foods contaminated with nerve agents. The route through which the poison enters the body largely determines the time required for the nerve agent to begin having an effect. It also influences the symptoms developed and, to some extent, the sequence of the different symptoms. Generally, poisoning takes place more rapidly when the agent is absorbed through the respiratory system than when it enters via other routes such as the skin. This is because the lungs contain numerous blood vessels which provide for rapid assimilation and transmission to the target organs. Nerve agents are more or less fat-soluble and can penetrate the outer layers of the skin. However, it takes some time before the poison reaches the deeper blood vessels. Consequently, the first symptoms may not appear until 20-30 minutes after the initial exposure. Chemically, nerve agents act by binding to an enzyme in the body of the victim, acetylcholinesterase, which inhibits this vital enzyme""s normal biological activity in the cholinergic nervous system. Acetylcholinesterase (xe2x80x9cAChExe2x80x9d) terminates nerve impulse transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine to acetate and choline. Organophosphate compounds such as insecticides and nerve agents inhibit AChE, which inhibition results in a build up of acetylcholine, thereby causing constant transmission of nerve signals.
Most recent research in the area of chemical and biological weapons has been focused on the detection and treatment of exposed individuals rather than the creation of new agents. Because the length of time that an individual is exposed to the agent can be determinative of the likelihood of successful treatment, rapid recognition that an exposure has occurred may mean the difference between life and death. Of course, this recognition/identification time includes not only the time required to perform the necessary diagnostic or chemical tests, but also the time required to move the victim or exposed item to a testing station or facility (or to move the testing unit to the victim, in some cases).
Certainly, there are any number of methods for detecting specific oganophosphate compounds in water or air. However, the methods suggested heretofore for are either too slow to make them useful for real time detection, or too bulky to be easily transported to a location near the front lines, where an attack would normally first be registered. For example, one common method of determining the presence of an OP compound is to measure the biochemical activity of acetylcholinesterase; if OP is present, the activity per enzyme molecule present decreases. However, this method is very slow and it might require days to get the sample to the lab and complete the tests. Additionally, even if conventional transportable units were fast enough to make them useful in real-time, they are too bulky to be distributed to and carried by every soldier which would be, of course, the best method of distribution. Further, most traditional methods of detecting nerve-type agents are designed to respond to one (or a few) specific compounds, which creates certain risks for in-field use, where the particular nerve gas variant might be different than expected
Heretofore, as is well known in the chemical and biological warfare arts, there has been a need for a method and apparatus that provides for rapid detection of nerve agents such as organophosphate compounds (xe2x80x9cOPxe2x80x9d compounds, hereinafter). This method should operate quickly and reliably to provide identification at the earliest possible moment, preferably in real-time or nearly so. It should work to detect these compounds in air or water and be portable and inexpensive enough to be issued to each individual who is at risk of exposure. Finally, it should a broad band detector which is responsive to a wide variety of OP compounds. Accordingly, it should now be recognized, as was recognized by the present inventor, that there exists, and has existed for some time, a very real need for an invention that would address and solve the above-described problems.
Before proceeding to a description of the present invention, however, it should be noted and remembered that the description of the invention which follows, together with the accompanying drawings, should not be construed as limiting the invention to the examples (or preferred embodiments) shown and described. This is so because those skilled in the art to which the invention pertains will be able to devise other forms of this invention within the ambit of the appended claims.
According to a first preferred aspect of the instant invention, there is provided a method for detecting the presence of nerve agents, OP compounds, and other molecules, including pesticides by detecting an alteration of the electron configuration and spectral properties of porphyrins and porphyrin surfaces and, recognizing the changes in protein conformation which are evidenced by alteration of porphyrins or other colorimetric indicators complexed with the protein. In brief, this embodiment of the instant invention detects an analyte by measuring the conformational change of its specific binding protein.
The instant invention is founded on the observation that complexes of protein with colorimetric compounds can be used to detect the presence of very low concentrations of hazardous or chemical warfare agents. Changes in the spectrum of a properly chosen colorimetric compound can be used as a xe2x80x9creal-timexe2x80x9d indicator to detect the presence of a broad range of dangerous substances such as nerve agents, organophosphates, and other chemical warfare agents. By way of general explanation and according to a first preferred embodiment, it is well known, and will be further discussed in the following narrative, that the electron distribution in a colorimetric compound is altered by its immediate environment. Changes in electron distribution result in corresponding changes in the spectrum of the colorimetric indicator. Thus, an indicator for use in detecting hazardous compounds may be created by monitoring specific lights wavelengths in the spectrum of a colorimetric compound of choice. Further, because of the multiplicity of absorbance bands in various of these indicators, unique spectral xe2x80x9csignaturesxe2x80x9d may be developed for use in subsequent detection.
Measurements of the optical changes in the colorimetric indicator are preferably made using both absorbance and fluorescence spectroscopy in the visible (400-800 nm) region. However, rather than attempt to directly sense changes in the spectrum of the indicator, difference spectra are preferably used instead, a difference spectrum being defined to be the spectrum of the indicator following exposure to an analyte minus the spectrum of the indicator prior to exposure. Wavelength shifts as small as 1-2 nm and absorbance changes down to or below 0.005A can be identified using the difference spectrum, thereby making it possible to identify over 7700 different analytes and quantify their concentration levels down to the 10xe2x88x929 M range.
Binding of substrate as well as inhibitors of enzymes may induce conformational changes in the enzyme. As is described hereinafter, changes in protein conformation induced by a substrate/inhibitor can be detected by porphyrins. More particularly, the change in conformation of acetylcholine esterase (the principal target for nerve agents and pesticides) upon binding of inhibitors is detected by colorimetric indicators such as porphyrins. Enzymes can be immobilized and complexed with porphyrins to make a solid-state monolayer reactive thin film sensor surface whose optical properties can readily be detected.
Additionally, and according to another preferred embodiment, there is provided a method and apparatus for the detection of nerve agents, OP compounds, and other molecules, including pesticides, which utilizes a reversible competitive inhibitor in combination with changes in the spectrum of the detecting materials to create a real-time sensor. More specifically, according to a preferred embodiment, an AChE-based biosensor is created by binding to the AChE molecule a porphyrin which inhibits it and which reversibly binds at the active site where substrate and/or nerve gasses and other inhibitors will bind. One such preferred porphyrin is TPPS1 (i.e., monosulfonate tetraphenyl porphyrin). When such a biosensor is exposed to nerve agents, etc., the OP compound will displace the selected porphyrin, thereby resulting in a change in the spectral properties of the biosensor.
According another aspect of the instant invention, there is provided an apparatus for detecting materials such as nerve agents, pesticides, and OP compounds, which uses real-time measurement of the changing spectral characteristics of a substrate as an indication of the presence of these materials. More particularly, the instant apparatus monitors the changing optical spectrum of a specially prepared colorimetrically responsive surface which indicates the presence of materials such as organophosphates through changes in its spectrum. Broadly speaking, the instant apparatus consists of a light source (preferably emitting light in the 400 nm to 800 nm range); a colorimetrically responsive surface which is illuminated by the irradiating light source; and, an optically sensitive detector which is directed toward the illuminated surface. In the preferred embodiment, the light source/detector combination operates continuously so that changes in the absorptive properties of the detection surface are immediately identified. It should be noted that the instant invention can be made small enough to take the form of a badge or similar device that might be worn continuously by at-risk personnel, and this device might also incorporate some sort of warning mechanism to notify the wearer the instant that OP compounds are detected. However, the instant inventor additionally contemplates that the light source and detector might be maintained separately from the detection surface, with identifying tests being conducted at some central location such as a testing station or laboratory.
In another preferred detection scheme, a biochemically active solid-state layer of AChE is deposited on a microscope slide. After exposure to an unknown compound, the slide is read via spectroscopy, not by shining light onto the face of the slide, but rather preferably by xe2x80x9cinjectingxe2x80x9d light into the glass slide containing the AChE through an edge of the slide. The effect of such delivery is that the incident light beam is reflected internally off of each parallel surface of the slide. Additionally, as is well known to those of ordinary skill in the art, the internally reflected beam extends about xc2xc wavelength (e.g., 100-200 nm depending on the wavelength of the incident light) into the surrounding medium through the creation of evanescent light waves. The evanescent light waves penetrate the detector layer on the surface of the slide and interact with the chemicals present thereon. As described previously, the intensity of light that has interacted with the detecting layer is then examined at at least two different spectral wavelengths for evidence of the presence of an OP compound. Preferably, the light that is supplied to the preferred slide embodiment, and which is the source of the evanescent waves, will be delivered by way of optical fibers.
Among the many agents/analytes which are believed to be detectable by the instant invention are xe2x80x9csimulantsxe2x80x9d of chemical and biological warfare agents: DIMP, DMMP, MPA, malathion, parathion and tetracain to simulate organo-phosphate agents such as Sarin or VX; and imidazole, methionine, thiodiethanol, cysteine, and other sulfur-containing organic molecules to simulate mustards.
In summary, the primary objectives of the instant invention are three fold. First, to utilize the spectral changes of colorimetric indicators such as porphyrins to identify chemical/biological agents (or simulants thereof) at different concentrations. In the preferred embodiment, the colorimetric indicator will either be a porphyrin in solution or a porphyrin immobilized onto a solid surface for use in test aqueous samples and samples in air.
A second object of the instant invention is to exploit the conformational changes in acetylcholine esterase and related enzymes induced by binding of inhibitors, including nerve agents, through the monitoring of spectral changes of colorimetric indicators reflective of that change. A third object of the instant invention is to utilize changes in the spectrographic properties of a reversible competitive inhibitor as a detector for OP and similar compounds.
The foregoing has outlined in broad terms the more important features of the invention disclosed herein so that the detailed description that follows may be more clearly understood, and so that the contribution of the instant inventor to the art may be better appreciated. The instant invention is not to be limited in its application to the details of the construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. Rather, the invention is capable of other embodiments and of being practiced and carried out in various other ways not specifically enumerated herein. Finally, it should be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting, unless the specification specifically so limits the invention.